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242 Arnau et al.
4. Desalting is necessary to remove imidazole that would otherwise inhibit DAPase
activity during tag removal.
5. DAPase requires the presence of a reducing thiol group for activity. It is
thought that at physiological pH, cysteamine and its oxidized form cystamine
act as a hydrogen donor for the reduction of disulfides in the enzymes.
Therefore, it is recommended to use freshly prepared enzyme cocktails with
cysteamine. Similarly, pGAPase bound to IMAC requires activation using
cysteamine prior to running the sample and after binding of the enzyme to
IMAC.
6. Enzyme activation by cysteamine is performed in small volumes to reduce the
amount needed.
7. Tag sequences containing Asp or Glu can only be digested at acidic pH, while
sequences containing His require pH above 6. Therefore, His-tag sequences
containing Glu or Asp can only be processed at pH 6–6.5.
8. Especially for upscaling purposes, it is important to reduce the amount of enzyme
used for tag removal. One approach is to run the cleavage at 4°C overnight
(see Fig. 5).
9. If the target protein concentration is 1–2 mg/ml, then 1 unit pGAPase per mg
of protein is recommended. For lower concentrations of target protein, higher
amounts of pGAPase are required, for example, at 0.75 mg/ml, 2 units/mg
pGAPase should be used.
10. The flow rate has great impact in the degree of completion of pyroglutamyl
removal. It is therefore not recommended to use higher flow rates.
References
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of recombinant thioredoxin fused N-terminal proCNP: analysis of enterokinase
cleavage products reveals new enterokinase cleavage sites. Protein Expr. Purif. 41,
332–340.
3. He, M., Jin, L. and Austen B. (1993) Specificity of factor Xa in the cleavage of
fusion proteins. J. Protein Chem. 12, 1–5.
4. Pedersen, J., Lauritzen, C., Madsen, M.T. and Dahl, S.W. (1999) Removal of N-
terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases.
Protein Expr. Purif. 15, 389–400.
5. TAGZyme manual (2003). Available from Qiagen at http://www1.qiagen.com/
literature/handbooks/PDF/Protein/Purification/QXP_TAGZyme/1024037_HBQXPT
AGZyme_032003.pdf
6. Hirel, P.H., Schmitter, J.M., Dessen, P., Fayat, G. and Blanquet, S. (1989) Extent
of N-terminal methionine excision from Escherichia coli proteins is governed by
the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. U. S. A.
86, 8247–8251.